Eliminating genome-wide and transcriptome-wide off-target mutations by base editor

Conjugation of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications, but suffers from off-target (OT) mutations. By taking advantage of a cleavable deoxycytidine deaminase inhibitor (dCDI) domain, a transformer BE (tBE) system is developed to induce efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, tBE remains inactive at OT sites with the fusion of a cleavable dCDI, thus eliminating unintended mutations. Only when binding at on-target sites, tBE is transformed to cleave off the dCDI domain and catalyzes targeted deamination for precise base changes. After delivery into mice via a dual-AAV system, tBE created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9 level, which resulted in ~30-40% decrease of total cholesterol. Together, the development of tBE establishes a highly-precise base editing system and its in vivo efficacy envisions potential therapeutic applications. Examination of whole transcriptome-wide off-target (OT) mutations induced by the transformer base editor (tBE)

CRISPR-Cas9与胞苷脱氨酶（cytidine deaminases）结合可得到碱基编辑器（base editors, BEs），用于可编程的C→T碱基编辑，其在临床应用中具备潜力，但存在脱靶（off-target, OT）突变问题。研究人员借助可切割的脱氧胞苷脱氨酶抑制剂（cleavable deoxycytidine deaminase inhibitor, dCDI）结构域，开发了Transformer碱基编辑器（Transformer BE, tBE）系统，该系统仅产生背景水平的全基因组及全转录组脱靶突变，即可实现高效编辑。tBE通过融合可切割的dCDI结构域，在脱靶位点保持无活性状态，从而消除意外突变。仅当结合至靶位点时，tBE才会切割去除dCDI结构域，催化靶向脱氨反应以实现精准的碱基改变。通过双腺相关病毒（dual-AAV）系统将tBE递送至小鼠体内后，其可在Pcsk9基因中引入提前终止密码子，显著降低血清PCSK9水平，进而使总胆固醇水平降低约30%~40%。综上，tBE的开发构建了一套高精度碱基编辑系统，其体内功效展现了潜在的治疗应用前景。本研究对Transformer碱基编辑器（tBE）诱导的全转录组脱靶突变进行了检测。